Although social housing of mice generally is preferred, mice must be individually housed in some situations. In these cases, enhanced attention to environmental enrichment is encouraged, but few studies assess the wellbeing of mice provided various enrichments. In this study, we used female ICR mice to evaluate enrichment strategies that encouraged natural behaviors including foraging, exercise, sheltering, and socialization. After 3 mo of exposure to the assigned enrichment strategy, wellbeing was assessed by evaluating behavioral and physiologic differences between groups. The results suggested that the use of red-tinted igloos may decrease markers of mouse wellbeing. However, none of the selected strategies yielded measures of wellbeing indicating improvement as compared to individually housed mice with no enrichment (negative control). Furthermore, measures were not significantly different between paired mice and individually housed mice with no enrichment.

Many variables can influence animal behavior and physiology, potentially affecting scientific study outcomes. Laboratory and husbandry procedures—including handling, cage cleaning, injections, blood collection, and animal identification—may produce a multitude of effects. Previous studies have examined the effects of such procedures by making behavioral and physiologic measurements at specific time points; this approach can be disruptive and limits the frequency or duration of observations. Because these procedures can have both acute and long-term effects, the behavior and physiology of animals should be monitored continuously. We performed a retrospective data analysis on the effects of 2 routine procedures, animal identification and cage changing, on motion and breathing rates of mice continuously monitored in the home cage. Animal identification, specifically tail tattooing and ear tagging, as well as cage changing, produced distinct and reproducible postprocedural changes in spontaneous motion and breathing rate patterns. Behavioral and physiologic changes lasted approximately 2 d after tattooing or ear tagging and 2 to 4 d for cage changing. Furthermore, cage changes showed strain-, sex-, and time-of-day–dependent responses but not age-dependent differences. Finally, by reviewing data from a rodent model of multiple sclerosis as a retrospective case study, we documented that cage changing inadvertently affected experimental outcomes. In summary, we demonstrate how retrospective analysis of data collected continuously can provide high-throughput, meaningful, and longitudinal insights in to how animals respond to routine procedures.

Ear tagging is perceived as less painful or stressful than tattooing and therefore is generally considered less harmful or costly to welfare. However, ear tags are more difficult to read than tattoos and can fall out, and mice usually require restraint for the tag numbers to be read accurately. We assessed the welfare and scientific implications of tattooing by using a commercial device compared with restraint in a device versus ear tagging. Male and female BALB/c mice (n = 32) underwent procedures after 1 wk of tail or nonaversive (tunnel) handling to determine whether tunnel handling reduced anxiety. Pain was evaluated using both the Mouse Grimace Scale (MGS) and manual and automated behavior analyses; light–dark preference testing and voluntary interaction with the handler's hand were used to assess anxiety. Tail inflammation after tattooing was quantified using bioluminescent imaging, and ear tag and tattoo misidentification rates were estimated from volunteer staff records. Tunnel handling reduced anxiety compared with tail handling. According to the MGS, tattooing was not more painful than ear tagging but caused significant tail inflammation and more agitation and anxiety. However, all tattoos were read correctly without handling, whereas all ear tagged mice needed restraint, and at least 25% of the tag codes were misread. Handling stress together with identification errors at this rate represent potentially serious concerns regarding the scientific integrity of data from studies using ear tagging. These concerns are unlikely to arise with tattooing. Although tattooing was stressful, so were restraint and ear tagging. However, considering the other major advantages of tattooing, the total costs associated with tattooing were not substantially greater than for ear tagging.

This study compared a synthetic bedding substrate (SBS), which has the potential to be a particulate-free animal bedding system, with the standard woodchip bedding. The objective was to demonstrate that the SBS is habitable for mice and reduces particulates to levels that would not contaminate the eye or potentially induce ocular (corneal) injury. Newly weaned mice were placed in either standard woodchip bedding or SBS. All mice were monitored regarding overall health (appearance, food and water intake, natural behavior, clinical signs, and provoked behavior) to verify their ability to adjust to the bedding. At 8 to 10 wk of age, the mice underwent slit-lamp evaluation for ocular (corneal) abnormalities. Results showed significant differences in body weight and overall health between bedding groups. The incidence of ocular abnormalities did not differ significantly between groups. We conclude that, without modifications and more testing, SBS is not a favorable bedding for mice, and results were inconclusive regarding its use as a bedding to preclude ocular contamination.

Chimpanzees demand specialized housing and care and the highest degree of attention to animal welfare. The current project used a survey method to collate information on chimpanzee housing and behavioral indices of welfare across all 6 of the chimpanzee research facilities in the United States. Data were compiled on 701 chimpanzees ranging from 2 to 62 y old (mean age, 26.0 y). All chimpanzees except for one were socially housed; the median group size was 7 animals, and group sizes ranged from 1 to 14. All of the subjects had access to outdoor spaces each day. Daily access to a natural substrate in the chimpanzee's enclosure was available for 63.8% of the subjects. Overall, 94.1% of the chimpanzees used tools to acquire food, 48.1% built nests, 75.8% copulated, and 83.3% initiated grooming bouts. The following atypical behaviors were reported most often: rocking (13.0%), coprophagy (10.0%), and stereotyped behaviors other than rocking (9.4%). There was widespread evi- dence of positive animal training techniques, with nearly all (97.7%) subjects reported to generally voluntarily cooperate with shifting in their enclosure, and 72.2% were reported to present for an injection of anesthetic. We include some comparison between these findings and data describing zoo-housed chimpanzees. In addition, we discuss survey findings in reference to recommendations made by the NIH Working Group on the Use of Chimpanzees in NIH-supported Research. The current survey assessed a larger sample of chimpanzees living under human care than has been published previously. This broad analysis can help to guide future improvements in behavioral management to address behavioral problems or deficits.

Rodent grimace scales facilitate assessment of ongoing pain. Reported rater training using these scales varies considerably and may contribute to the observed variability in interrater reliability. This study evaluated the effect of training on interrater reliability with the Rat Grimace Scale (RGS). Two training sets (42 and 150 images) were prepared from acute pain models. Four trainee raters progressed through 2 rounds of training, scoring 42 images (set 1) followed by 150 images (set 2a). After each round, trainees reviewed the RGS and any problematic images with an experienced rater. The 150 images were then rescored (set 2b). Four years later, trainees rescored the 150 images (set 2c). A second group of raters (no-training group) scored the same image sets without review with the experienced rater. Inter- and intrarater reliability were evaluated by using the intraclass correlation coefficient (ICC), and ICC values were compared by using the Feldt test. In the trainee group, interrater reliability increased from moderate to very good between sets 1 and 2b and increased between sets 2a and 2b. Action units with the highest and lowest ICC at set 2b were orbital tightening and whiskers, respectively. In comparison to an experienced rater, the ICC for all trainees improved, ranging from 0.88 to 0.91 at set 2b. Four years later, very good interrater reliability was retained, and intrarater reliability was good or very good). The interrater reliability of the no-training group was moderate and did not improve from set 1 to set 2b. Training improved interrater reliability, with an associated reduction in 95%CI. In addition, training improved interrater reliability with an experienced rater, and performance was retained.

In this review, we describe the methods and technology used to measure intracage ammonia levels; the data were derived from 38 articles published since 1970. Ammonia concentration is commonly used as a surrogate for assessing environmental quality inside rodent cages. Data generated from this group of publications have been used to support new husbandry practices, determine the effect of ammonia on health, and establish the effectiveness of caging systems. Consequently, the data generated from these studies have a direct effect on animal welfare and therefore should demonstrate a high level of reproducibility. Obtaining reproducible results requires a critical understanding of the methodology and the technology used to collect ammonia concentration data. This review highlights the need for consistent methodology for measuring ammonia that considers the technology used to capture the data as well as the environmental parameters that affect ammonia concentrations, to facilitate the design of future studies.

Accidental exposure of our mice to bisphenol A (BPA) from damaged polycarbonate cages 20 y ago provided some of the first evidence of the harmful effects of exposure to this common chemical. Recently we found that housing mice in damaged polysulfone cages resulted in similar harmful effects due to exposure to bisphenol S (BPS). This problem was unexpected for 2 reasons. First, polysulfone is a far more chemically resistant polymer than polycarbonate. Second, BPS is not a component in the manufacture of polysulfone. We report here our efforts to verify the source of the BPS and eliminate the exposure. Our analysis of new polysulfone caging materials confirmed that BPS is a breakdown product of damaged polysulfone plastic. Furthermore, we found that BPS can cross-contaminate new or undamaged cages in facilities that process damaged caging materials. Neither the use of disposable cages nor replacement of caging materials used solely for our colony was sufficient to eliminate exposure effects. Only the replacement of all cages and water bottles in the facility corrected the problem and allowed us to resume our studies. Taken together, our previous and current findings underscore the concern that chemicals from plastics are harmful environmental contaminants for both humans and animals. Furthermore, our results provide strong evidence that the presence of damaged plastic in a facility may be sufficient to affect research results and, by extension, animal health.

Rodent sentinel screening for adventitious pathogens is an integral part of many biomedical research institutes and universities that use rodents in research. Typical screening programs involving live sentinel animals typically purchase young SPF sentinel animals that are sampled and replaced quarterly. Previous reports suggest that mice as old as 6 mo are effective sentinels for various agents. In efforts to reduce the number of animals used in our sentinel program, we wanted to investigate the possibility of keeping sentinel animals inhouse for 12 mo at a time. We exposed mice (age, 40 to 48 wk) to murine norovirus (MNV) to test whether they could reliably produce detectable levels of antibodies (similar to younger mice) to this adventitious pathogen. Mice first exposed to MNV at 40 to 48 wk of age seroconverted to MNV after both direct inoculation (through gavage) and indirect exposure (from soiled-bedding transfer) at the same or greater frequency than mice first exposed at 8 to 12 wk of age. These findings indicate that, at least for MNV, sentinel residence time can be extended from 3 to 12 mo without compromising the reliability of seroconversion, thus ultimately reducing sentinel animal numbers. This practice, combined with nonanimal testing modalities (for example, exhaust duct sampling), can increase the sensitivity and specificity of rodent surveillance programs and minimize the use of live animals.

The precise identification of rodent Pasteurellaceae is known to be highly challenging. An unknown strain of Pasteurellaceae appeared and rapidly spread throughout our animal facilities. Standard microbiology, combined with biochemical analysis, suggested that the bacteria strain was Rodentibacter pneumotropicus or R. heylii. We submitted samples of the unknown bacteria and known isolates of R. pneumotropicus, R. heylii, and Muribacter muris, to 2 service laboratories that provide animal health monitoring. Results of microbiology tests performed by both laboratories, species-specific PCR analysis performed by one laboratory, and independent 16S rRNA gene sequencing yielded identical identification of the unknown bacteria as Pasteurellaceae (Pasteurella spp.) and not R. pneumotropicus or R. heylii. In contrast, the similarly intended PCR assay performed by the other laboratory identified the bacteria as R. heylii. Careful evaluation of all of the results led us to conclude that the correct identification of the bacteria is Pasteurellaceae. From our experience, we recommend that a combination of several methods should be used to achieve correct identification of rodent Pasteurellaceae. Specifically, we advise that all primer sets used should be disclosed when reporting PCR test results, including in health reports provided by service laboratories and animal vendors. Careful, correct, and informative health monitoring reports are most beneficial to animal researchers and caretakers who might encounter the presence and effects of rodent Pasteurellaceae.

Exposing immunodeficient mice to opportunistic microbes introduces risks of data variability, morbidity, mortality, and the invalidation of studies involving unique human reagents, including the loss of primary human hematopoietic cells, patient-derived xenografts, and experimental therapeutics. The prevalence of 15 opportunistic microbes in a murine research facility was determined by yearlong PCR-based murine and IVC equipment surveillance comprising 1738 specimens. Of the 8 microbes detected, 3 organisms— Staphylococcus xylosus, Proteus mirabilis, and Pasteurella pneumotropica biotype Heyl—were most prevalent in both murine and IVC exhaust plenum specimens. Overall, the 8 detectable microbes were more readily PCR-detectable in IVC exhaust airways than in murine specimens, supporting the utility of PCR testing of IVC exhaust airways as a component of immunodeficient murine health surveillance. Vaporized hydrogen peroxide (VHP) exposure of IVC equipment left unassembled (that is, in a 'static-open' configuration) did not eliminate PCR detectable evidence of microbes. In contrast, VHP exposure of IVC equipment assembled 'active-closed' eliminated PCR-detectable evidence of all microbes. Ensuring data integrity and maintaining a topographically complex immunodeficient murine research environment is facilitated by knowing the prevalent opportunistic microbes to be monitored and by implementing a PCR-validated method of facility decontamination that mitigates opportunistic microbes and the risk of invalidation of studies involving immunodeficient mice.

This study compared alfaxalone, alone and in combination with other medications, for sedative and anesthetic properties after intramuscular administration in New Zealand white rabbits. In the main portion of the study, 6 female rabbits were assigned to 5 treatment regimens in a blinded crossover design. Alfaxalone (6 mg/kg IM) was administered alone and in combination with each of the following: 0.3 mg/kg butorphanol; 1 mg/kg midazolam; 0.2 mg/kg dexmedetomidine; and both 0.3 mg/kg butorphanol and 0.2 mg/kg dexmedetomidine. An additional 6 rabbits received 0.2 mg/kg dexmedetomidine for comparison. The median time to onset of recumbency ranged from 2.0 to 5.5 min, with times significantly shorter for animals that received alfaxalone with either midazolam or dexmedetomidine than for those given dexmedetomidine only. Duration of sedation (mean ± 1 SD) was: alfaxalone only, 40 ± 7.3 min; alfaxalone with butorphanol, 47.8 ± 9.9 min; alfaxalone with midazolam, 65.2 ± 6.5 min; alfaxalone with dexmedetomidine, 157.5 ± 22.4 min; alfaxalone with butorphanol and dexmedetomidine, 157.7 ± 22.3 min, and dexmedetomidine only, 93.7 ± 11.9 min. Response to noxious stimuli was absent in 2 of the rabbits given dexmedetomidine only, 4 of those given alfaxalone with dexmedetomidine, and all 6 of the animals dosed with alfaxalone, butorphanol, and dexmedetomidine; this last group displayed the longest absence of a toe-pinch response (57 ± 3 min).

Regional anesthesia is a commonly used adjunct to orofacial dental and surgical procedures in companion animals and humans. However, appropriate techniques for anesthetizing branches of the mandibular and maxillary nerves have not been described for rhesus monkeys. Skulls of 3 adult rhesus monkeys were examined to identify relevant foramina, establish appropriate landmarks for injection, and estimate injection angles and depth. Cadaver heads of 7 adult rhesus monkeys (4 male, 3 female) were then injected with thiazine dye to demonstrate correct placement of solution to immerse specific branches of the mandibular and maxillary nerves. Different volumes of dye were injected on each side of each head to visualize area of diffusion, and to estimate the minimum volume needed to saturate the area of interest. After injection, the heads were dissected to expose the relevant nerves and skull foramina. We describe techniques for blocking the maxillary nerve as well as its branches: the greater palatine nerve, nasopalatine nerve, and infraorbital nerve. We also describe techniques for blocking branches of the mandibular nerve: inferior alveolar nerve, mental (or incisive) nerve, lingual nerve, and long buccal nerve. Local anesthesia for the mandibular and maxillary nerves can be accomplished in rhesus macaques and is a practical and efficient way to maximize animal welfare during potentially painful orofacial procedures.

This pharmacokinetic study was designed to determine the pharmacokinetics of enrofloxacin at 5 mg/kg when given to sea hares in their hemolymph. Enrofloxacin is a commonly used antimicrobial in veterinary medicine and potentially could be used to treat sea hares exposed to susceptible bacterial species. We individually identified 8 juvenile Aplysia californica and group housed them in an open seawater flow system at 14 to 18 °C; 2 served as untreated controls. The remaining 6 animals were injected into the hemocoel with 0.030 mL of 22.7 mg/mL enrofloxacin (average dose, 5 to 6 mg/kg). At each time point, 300 μL hemolymph was collected from the pedal hemolymph sinus and HPLC-analyzed for enrofloxacin and ciprofloxacin levels. Enrofloxacin was detected in all dosed animals, at an average peak concentration of 3 μg/mL in hemolymph, and remained in the body for 20.3 h with an average clearance of 0.19 μg × h/mL. No ciprofloxacin was detected in any Aplysia in this study. Hemocoel injection appears to be an effective way to administer enrofloxacin to Aplysia and reach clinically relevant concentrations. Enrofloxacin reached therapeutic target concentrations in A. californica when dosed according to the regimen described in the current report.

level and improve surgical outcomes. Recently, some institutions have approved the use of Press'n Seal cling film (CF; Glad Products, Oakland, CA) as a practical, cost-effective alternative to sterile drapes for rodent surgeries. The purpose of this study was to evaluate the sterility of CF by using ATP and replicate organism detection and counting (RODAC) plates. We tested 10 boxes of CF at days 0, 14, and 28 after opening the box and compared the results with traditional packaged sterile drapes. Our data indicated that CF ATP bioluminescence remained at or below 10 relative light units for 28 d after opening the box. In addition, RODAC plates had no growth for 70% of CF boxes at day 0, 100% at day 14, and 90% at day 28. The mean growth for the positive plates was 0.024 cfu/cm² sampled after contacting locations on the front and back of the CF. The results of this study support the use of CF as an acceptable alternative to traditional sterile drapes during rodent aseptic surgery.

Rabbits provide a unique challenge for routine endotracheal intubation in clinical practice because of various distinctive anatomic and physiologic features. Many previously proposed methods for endotracheal intubation in rabbits are limited by several factors, including the needs for expensive equipment and high levels of technical expertise. We evaluated capnography for its effectiveness in assisting endotracheal intubation in rabbits. New Zealand white rabbits were divided into 3 groups of 5 animals. In the first 2 groups, mainstream (nondiverting) or sidestream (diverting) capnography (MC and SC groups, re- spectively) was used; in the third group (LS group), a laryngoscope with a size 00 Miller blade was used to guide endotracheal tube placement. Anesthesia was induced through intramuscular administration of ketamine (10 mg/kg), medetomidine (0.1 mg/kg), and midazolam (1 mg/kg) mixed in the same syringe prior to administration. Intubation time was defined from the point of opening the jaws to the completion of the first capnogram after intubation. Intubation was accomplished successfully in all animals in both capnography groups, but 2 rabbits in the laryngoscopy group could not be intubated. Intubation time was compared among groups was compared by using one-way ANOVA, and posthoc Bonferroni testing was applied to isolate significant differences between groups. The intubation time (mean ± 1 SD) was 46.4 ± 12.6 s in the MC group, 147.2 ± 44.2 s in the SC group, and 385.0 ± 114.1 in the LS group, with intubation time significantly differing among all groups. In conclusion, both mainstream and sidestream capnography-guided endotracheal intubation techniques were more effective and efficient than conventional laryngoscope-guided endotracheal intubation in rabbits. Furthermore, mainstream capnography was preferred over sidestream capnography because mainstream capnography resulted in significantly shorter intubation times.

Immunodeficient mice in multiple holding rooms presented with head tilt, circling, spinning when picked up by the tail, dehydration, and lethargy. Burkholderia gladioli, a plant pathogen, was identified as the causative agent. Environmental testing revealed the presence of B. gladioli within the automatic watering system, water bottles, and sipper tubes. Here we describe steps taken to reduce the presence of this organism within the automatic watering system and water bottles. Facilities housing immunodeficient mice should take measures to minimize the accumulation of biofilm within their water-supply systems.